超薄聚二甲基硅氧烷微氟化片在保水作用下固定化活线虫个体
Summary
建立了一系列固定化方法, 允许使用最近开发的具有水保留的超薄聚二甲基硅氧烷微流体芯片对活的虫进行定向照射。这种新颖的片上固定也足以进行成像观测。详细说明了该芯片的处理和应用实例。
Abstract
辐射被广泛用于生物应用和离子束育种, 在这些方法中, 微束辐射是识别活生物体中放射性敏感位点的有力手段。本文介绍了为线虫活体个体的靶向微束照射而开发的一系列片上固定方法。值得注意的是, 我们以前开发的聚二甲基硅氧烷 (PDMS) 微流体芯片的治疗方法是在不需要麻醉的情况下固定线虫个体的。这种芯片, 被称为蠕虫片, 是弹性, 允许微流体通道被扩大, 弹性允许动物被轻轻包围。此外, 由于 PDMS 的自吸附能力, 动物可以通过用薄膜覆盖蠕虫片的表面, 在其中不被推入通道进行围护结构, 从而将动物密封在通道中。通过翻拍封面片, 我们可以很容易地收集动物。此外, 蠕虫片显示水的保留, 并允许线虫人在活的条件下进行长时间的微观观察。此外, 板材厚度仅为 300μm, 允许碳离子等重离子穿过封闭动物的片状物, 从而可检测离子粒子并准确测量所施加的辐射剂量。由于用于围住动物的封面膜的选择对成功的长期固定非常重要, 我们对合适的封面片进行了选择, 并在一些电影中展示了推荐的封面电影。作为芯片的应用实例, 我们介绍了动物包围蜗杆片微流体通道的肌肉活动的成像观测, 以及微束照射。这些例子表明, 蠕虫板极大地扩大了生物实验的可能性。
Introduction
辐射, 包括 x 射线、伽马射线和重离子束, 广泛用于生物应用, 如癌症诊断和治疗, 以及离子束育种。目前, 许多研究和技术发展的重点是辐射的影响1、2、3。微束照射是识别生物体中放射性敏感位点的有力手段 4.美国量子与放射科学与技术研究所 (QST-Takasaki) 高崎高级辐射研究所 (QST-Takasaki) 一直在开发一种利用重离子在显微镜下照射单个细胞的技术微束5, 并建立了方法, 使目标微束照射的几个模型动物, 如线虫线虫4,6, 蚕7和oryzias纬度(日本美达卡)8。线虫线虫的靶向微束照射可以有效地击倒特定区域, 如头部区域的神经环, 从而有助于确定这些系统在运动等过程中的作用。
开发了一种不需要麻醉的线虫人片上固定化的方法, 以允许微束照射 4.此外, 为了改进上一项研究4中使用的微流体芯片, 我们最近开发了可湿性、离子渗透、聚二甲基硅氧烷 (PDMS) 微流体芯片, 称为蠕虫片 (见材料表), 用于固定线虫人9。这些包括超薄软片 (厚度 = 300μm; 宽度 = 15 毫米; 长度 = 15 毫米), 表面有多个 (20 或 25) 直微流体通道 (深度 = 70μm; 宽度 = 60μm 或 50μm; 长度 = 8 mm) (图 1a-d)。微流体通道是开放的, 允许多个动物同时被封闭在其中 (图 1e)。这些薄片具有弹性, 可以扩大微流体通道 (约 10%,图 1f), 弹性允许动物轻轻包裹。此外, 由于 PDMS 的自吸附能力, 动物可以通过用薄膜覆盖蠕虫片的表面, 在其中不被推入通道进行围护结构, 从而将动物密封在通道中。通过翻拍封面片, 我们可以很容易地收集动物。
这些通道在被封闭或收集时不会伤害虫子。此外, 板材是由 PDMS, 这基本上是疏水, 但水的保持可以通过赋予亲水性的材料。水的保留性和厚度是蜗杆板的良好特性。保水能力可防止动物在长时间固定后脱水, 并可进行长期观察。
此外, 如前所述, 板材只有300μm 厚, 允许重离子, 如碳离子 (水的范围约为1毫米) 通过片状包围动物。这样就可以检测离子粒子, 准确测量所施加的辐射剂量。此外, 蠕虫板可以重复使用, 因此是经济的。用传统的注射方法, 封闭的动物有时会死亡, 不能被带出通道;他们的蛋也会堵塞频道。这使得芯片无法使用。因此, 芯片基本上是一次性的, 成本效益比率很低。
本文详细介绍了用蠕虫片对活体线虫进行片上固定化的一系列方法。通过对片上固定3小时后动物的运动, 对合适的覆盖膜进行了评价。此外, 我们还展示了用于成像观测和微束照射的片上固定化示例。
Protocol
Representative Results
Discussion
在带电条件下, 使用可湿性 PDMS 微流体芯片对线虫进行片上固定, 使多种动物能够有效地进行有针对性的微束照射。易于操作和防止干燥的特点使该系统不仅适用于微束照射, 而且适用于几种行为检测。这些蠕虫单已经商业化, 可以很容易地获得。传统的微流体芯片, 如嗅觉芯片, 与诸如在封闭的微流体通道中堵塞动物和鸡蛋等问题有关, 使它们难以采集动物, 因此, 这种芯片往往是一次性的, ?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢 Atsushi Higashitani 博士就线虫和 Yuya hari 博士、Yasuhiko Yokota 和 Kobayashi 康彦的治疗提出的善意建议, 并进行了宝贵的讨论。作者感谢 Caenorhabditis 遗传中心提供了线虫和大肠杆菌菌株。我们感谢 QST-Takasaki 的 TIARA 回旋加速器的机组人员对辐照实验的善意帮助。我们感谢苏珊·弗尼斯博士编辑了这份手稿的草稿。这项研究得到了从 JPS 到 m. s. 的 KAKENHI (赠款号码 JAK15K11921 和 J18K1839) 的部分支持。
Materials
| C. elegans wild-type strain | Caenorhabditis Genetics Center (CGC) , Minnesota, USA | N2 | Wild-type C. elegans strain generally used in this study |
| C. elegans unc-119(e2498) III mutant strain | Caenorhabditis Genetics Center (CGC) , Minnesota, USA | CB4845 | C. elegans strain only employed as an example of mutants with abnormal body shape |
| C. elegans transgenic strain HBR4 | Caenorhabditis Genetics Center (CGC) , Minnesota, USA | HBR4 | The genotype of this transgenic C. elegans strain is HBR4:goeIs3[pmyo-3::GCamP3.35:: unc-54–3’utr, unc-119(+)]V. This strain was only employed for imaging observation. |
| E. coli strain | Caenorhabditis Genetics Center (CGC) , Minnesota, USA | OP50 | E. coli strain used as food for C. elegans |
| Worm Sheet IR (50/60) | Biocosm, Inc., Hyogo, Japan | BCM17-0001 | Microfluidic chip with 25 straight 50/60-µm width channels used in all experiments and observation in this paper |
| Worm Sheet 60 | Biocosm, Inc., Hyogo, Japan | BCM18-0001 | Microfluidic chip with 20 straight 60 µm-width channels. This is sitable for adults 3-5 days after hatching at 20°C. |
| Worm Sheet 50 | Biocosm, Inc., Hyogo, Japan | BCM18-0002 | Microfluidic chip with 20 straight 50 µm-width channels. This is sitable for youg adults ~3 days after hatching at 20°C. |
| MICRO COVER GLASS | MATSUNAMI GLASS IND. LTD. | C030401 | Cover glass (thickness: 130-170 µm) used in locomotion assays in Protocol 3 |
| Polystyrene Film | Biocosm, Inc., Hyogo, Japan | BCM18-0001/ BCM18-0002 | Bundled items of Worm Sheets. PS filim (thickness: ~130 µm) used in locomotion assays in Protocol 3. |
| Polyester Film Lumirror | TORAY INDUSTRIES, INC., Tokyo, Japan | Lumirror T60 (t 125 µm) | PET filim (thickness: 125 µm) used in locomotion assays in Protocol 3 |
| IWAKI 60 mm/non-treated dish | AGC Techno Glass Co., Ltd., Shizuoka, Japan). | 1010-060 | Non-treated dish used in incuvation of C. elegans in Protocol 1 |
| IWAKI 35 mm/non-treated dish | AGC Techno Glass Co., Ltd., Shizuoka, Japan). | 1010-035 | Non-treated dish used in locomotion assays in Protocol 3 |
| Milli-Q | Merck, France | Ultrapure water | |
| Kimwipe S-200 | Nippon Paper Crecia Co., Ltd., Tokyo, Japan | 62020 | 120 mm x 215 mm; 200 sheets/ box |
| WormStuff Worm Pick | Genesee Scientific Corporation, CA, USA) | 59-AWP | Platina picker specilized for picking up C. elegans |
| Research Stereo Microscope System | OLYMPUS CORPORATION, Tokyo, Japan | SZX16 | Micriscope used in all experiments and observation in this paper |
| Motorized Focus Stand for SZX16 | OLYMPUS CORPORATION, Tokyo, Japan | SZX2-ILLB | This was used for bright field observation in Protocol 3-8. |
| Objective Lens (×1) | OLYMPUS CORPORATION, Tokyo, Japan | SDFPLAPO1×PF | NA: 0.15; W.D.: 60 mm. This lends was used for bright field observation in Protocol 3-8. |
| Objective Lens (×2) | OLYMPUS CORPORATION, Tokyo, Japan | SDFPLAPO2XPFC | NA: 0.3; W.D.: 20 mm. This lends was used for imaging observations. |
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